1. Technical Field
The present invention generally relates to gene expression and more specifically to immediate early genes in the brain and polypeptides encoded by such immediate early genes.
2. Background Information
An immediate early gene (IEG) is a gene whose expression is rapidly increased immediately following a stimulus. For example, genes expressed by neurons that exhibit a rapid increase in expression immediately following neuronal stimulation are neuronal IEGs. Such neuronal IEGs have been found to encode a wide variety of polypeptides including transcription factors, cytoskeletal polypeptides, growth factors, and metabolic enzymes as well as polypeptides involved in signal transduction. The identification of neuronal IEGs and the polypeptides they encode provides important information about the function of neurons in, for example, learning, memory, synaptic transmission, tolerance, and neuronal plasticity.
The present invention involves methods and materials related to IEGs. Specifically, the invention provides isolated IEG nucleic acid sequences, cells that contain isolated IEG nucleic acid, substantially pure polypeptides encoded by IEG nucleic acid, and antibodies having specific binding affinity for a polypeptide encoded by IEG nucleic acid. In addition, the invention provides cDNA libraries enriched for IEG cDNAs, isolated nucleic acid derived from such cDNA libraries, and methods for treating conditions related to a deficiency in a neuron""s IEG responsiveness to a stimulus.
In one aspect, the invention features an isolated nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. The isolated nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. The hybridization conditions can be moderately or highly stringent hybridization conditions.
In another embodiment, the invention features an isolated nucleic acid having a nucleic acid sequence that encodes an amino acid sequence at least five amino acids in length. The amino acid sequence contains at least three different amino acid residues, and is identical to a contiguous portion of sequence set forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62.
Another embodiment of the invention features an isolated nucleic acid having a nucleic acid sequence at least 60 percent identical to the sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
Another embodiment of the invention features an isolated nucleic acid having a nucleic acid sequence that encodes an amino acid sequence at least 60 percent identical to the sequence set forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62.
Another embodiment of the invention features an isolated nucleic acid having a nucleic acid sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
In another aspect, the invention features a substantially pure polypeptide having an amino acid sequence encoded by a nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. The nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
In another embodiment, the invention features a substantially pure polypeptide having an amino acid sequence as set forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62.
Another embodiment of the invention features a substantially pure polypeptide having an amino acid sequence at least 60 percent identical to the sequence set forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62.
Another embodiment of the invention features a substantially pure polypeptide having an amino acid sequence at least five amino acids in length. The amino acid sequence contains at least three different amino acid residues, and is identical to a contiguous stretch of sequence set forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62.
Another aspect of the invention features a host cell (e.g., a eukaryotic or prokaryotic cell) containing an isolated nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. The isolated nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
Another aspect of the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity for an amino acid sequence encoded by a nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. The nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
Another aspect of the invention features a cDNA library having a plurality of clones with each clone having a cDNA insert. In addition, at least about 15 percent (e.g., at least about 20 or 25 percent) of the clones have cDNA derived from immediate early genes (e.g., immediate early genes responsive to a maximal electroconvulsive seizure). The cDNA library can be a subtracted cDNA library. For example, the subtracted cDNA library can be the IEG-Reg or IEG-Lg cDNA library.
Another aspect of the invention features an isolated nucleic acid derived from a cDNA library. The cDNA library has a plurality of clones with each clone having a cDNA insert. In addition, at least about 15 percent of the clones have cDNA derived from immediate early genes. The isolated nucleic acid can have a nucleic acid sequence of an immediate early gene.
Another aspect of the invention features a method of obtaining immediate early gene nucleic acid. The method includes providing a cDNA library having a plurality of clones with each clone having a cDNA insert. In addition, at least about 15 percent of the clones have cDNA derived from immediate early genes. The method also includes contacting at least a portion of the cDNA library with a probe containing at least one nucleic acid having a nucleic acid sequence derived from an immediate early gene, and selecting a member of the plurality of clones based on the hybridization of the at least one nucleic acid to the member under hybridization conditions.
Another aspect of the invention features a method of treating an animal (e.g., human) having a deficiency in a neuron""s immediate early gene responsiveness to a stimulus. The method includes administering a nucleic acid to the animal such that the effect of the deficiency is minimized. The nucleic acid has at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. In addition, the nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. The deficiency can include a reduced level of expression of an immediate early gene. In addition, the stimulus can influence learning or memory. For example, the stimulus can include a maximal electroconvulsive seizure.
In another embodiment, the invention features a method of treating an animal (e.g., human) having a deficiency in a neuron""s immediate early gene responsiveness to a stimulus. The method includes administering a therapeutically effective amount of a substantially pure polypeptide to the animal such that the effect of the deficiency is minimized. The polypeptide contains an amino acid sequence encoded by a nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. The nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
Another embodiment of the invention features a method of treating an animal (e.g., human) having a deficiency in a neuron""s immediate early gene responsiveness to a stimulus. The method includes administering an effective amount of cells to the animal such that the effect of the deficiency is minimized. The cells contain a nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. In addition, the nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22,23,24,25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
Another embodiment of the invention features a method of treating an animal (e.g., human) having a deficiency in a neuron""s immediate early gene responsiveness to a stimulus. The method includes administering a therapeutically effective of antibodies to the animal such that the effect of the deficiency is minimized. The antibodies have specific binding affinity for an amino acid sequence encoded by a nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. The nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. The deficiency can include an elevated level of expression of an immediate early gene.
Another aspect of the invention features a method of identifying a compound that modulates immediate early gene expression. The method includes contacting a test compound with an immediate early gene nucleic acid, and determining whether the test compound effects the expression of the immediate early gene nucleic acid. The presence of an effect indicates that the test compound is a compound that modulates immediate early gene expression. The immediate early gene nucleic acid can contain a nucleic acid sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. The effect can be a reduction or increase in the expression of the immediate early gene nucleic acid.
In another embodiment, the invention features a method of identifying a compound that modulates immediate early gene polypeptide activity. The method includes contacting a test compound with an immediate early gene polypeptide, and determining whether the test compound effects the activity of the immediate early gene polypeptide. The presence of an effect indicates that the test compound is a compound that modulates immediate early gene polypeptide activity. The immediate early gene polypeptide can contain an amino acid sequence encoded by a nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base. The nucleic acid is at least 12 bases in length, and hybridizes to the sense or antisense strand of a second nucleic acid under hybridization conditions. The second nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. Alternatively, the immediate early gene polypeptide can contain an amino acid sequence as set forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62. The effect can be a reduction or increase in the activity of the immediate early gene polypeptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The present invention provides methods and materials related to IEGs. Specifically, the invention provides isolated IEG nucleic acid, cells that contain isolated IEG nucleic acid, substantially pure polypeptides encoded by IEG nucleic acid, and antibodies having specific binding affinity for a polypeptide encoded by IEG nucleic acid. In addition, the invention provides cDNA libraries enriched for IEG cDNAs, isolated nucleic acid derived from such cDNA libraries, and methods for treating conditions related to a deficiency in a neuron""s IEG responsiveness to a stimulus.
The present invention is based on the discovery of nucleic acid clones for many different neuronal IEGs. Specifically, nucleic acid clones for different neuronal IEGs were isolated and identified based on the ability of each IEG to rapidly increase expression upon seizure induction by a maximal electroconvulsive seizure (MECS) method (Cole et al., J. Neurochem. 55:1920-1927 (1990)). It is important to note that MECS induction can be considered a model to study long-term plasticity relevant to learning and memory since it is known that a single MBCS can produce extremely robust and long lived potentiation of synaptic contacts in the hippocampus and block spatial learning (Barnes et al., J. Neurosci. 14:5793-5806 (1994)). Thus, MECS-responsive IEGs can influence neuronal activities involved in brain functions such as learning and memory. Moreover, the isolation and identification of IEG nucleic acid not only provides research scientists with information about neuronal activity and gene regulation but also provides methods and materials that can be used to manipulate brain function.
Each isolated IEG nucleic acid described herein can be used to produce a polypeptide. In addition, each IEG nucleic acid can be used to identify cells that are responsive to MECS induction. For example, an IEG nucleic acid can be labeled and used as a probe for in situ hybridization analysis. Clearly, having the ability to identify MECS-responsive cells provides one with the ability to isolate or monitor specific brain regions that are involved in learning. Further, any of the isolated partial IEG nucleic acid sequences can be used to obtain a full-length clone that encodes an IEG polypeptide. For example, a fragment from an isolated IEG nucleic acid can be radioactively labeled and used to screen a library such that a full-length clone is obtained.
Cells containing isolated IEG nucleic acid can be used to maintain or propagate the isolated IEG nucleic acid. In addition, such cells can be used to produce large quantities of polypeptides that are encoded by isolated IEG nucleic acid. Further, cells containing isolated IEG nucleic acid can be used to generate virus particles containing the isolated IEG nucleic acid. Such virus particles can be used in vitro or in vivo to provide other cells with the isolated IEG nucleic acid. The polypeptides encoded by IEG nucleic acid can be used as immunogens to produce antibodies. Such antibodies can be used to identify MECS-responsive cells, monitor the level of polypeptide expression following MECS induction, and isolate polypeptides directly from animal tissue.
cDNA libraries enriched for IEG cDNAs can be used to isolate novel IEG cDNA. Clearly, the isolation of novel IEG cDNAs is important to further the understanding of brain function. In addition, isolated nucleic acid derived from such cDNA libraries can be used to produce polypeptides as well as identify cells that are responsive to a stimulus such as MECS induction.
It is important to note that isolated IEG nucleic acid, cells containing isolated IEG nucleic acid, substantially pure IEG polypeptides, and anti-IEG polypeptide antibodies can be used to treat conditions associated with a deficiency in a neuron""s ability to express IEGs in response to a stimulus such as MECS. A condition associated with a deficiency in a neuron""s IEG responsiveness to a stimulus is any physiological condition characterized as having a lack of a normal level of responsiveness. For example, when a deficiency in a neuron""s responsiveness to MECS is characterized as a non- or under-expression of a particular IEG polypeptide by that neuron, the organism having the condition can be treated with isolated IEG nucleic acid, cells containing isolated IEG nucleic acid, or substantially pure IEG polypeptides such that the effect of the deficiency is minimized. Alternatively, when a deficiency in a neuron""s responsiveness to MECS is characterized as an over-expression of a particular IEG polypeptide by that neuron, the organism having the condition can be treated with anti-IEG polypeptide antibodies or the anti-sense strand of an isolated IEG nucleic acid such that the effect of the deficiency is minimized.
In addition, isolated IEG nucleic acid, cells containing isolated IEG nucleic acid, substantially pure IEG polypeptides, and anti-IEG polypeptide antibodies can be used to identify pharmaceutical compounds that can be used to treat diseases such as epilepsy, age-dependent memory decline, stroke, and drug addiction. For example, a compound that modulates IEG nucleic acid expression or IEG polypeptide activity can be identified by contacting a test compound with either the IEG nucleic acid or polypeptide, and determining whether the test compound effects expression or activity.
The term xe2x80x9cnucleic acidxe2x80x9d as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
The term xe2x80x9cisolatedxe2x80x9d as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.
The term xe2x80x9cisolatedxe2x80x9d also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include, a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.
It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.
Any isolated nucleic acid having a nucleic acid sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 is within the scope of the invention. For convenience, these nucleic acid sequences will be referred to collectively as the IEG nucleic acid group. In addition, any isolated nucleic acid having a nucleic acid sequence at least about 60 percent identical (e.g., at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent identical) to a sequence set forth in the IEG nucleic acid group is within the scope of the invention. For the purpose of this invention, the percent identity between a sequence set forth in the IEG nucleic acid group (designated a template sequence) and any other nucleic acid sequence is calculated as follows. First, the two nucleic acid sequences are aligned using the MEGALIGN(copyright) (DNASTAR, Madison, Wis., 1997) sequence alignment software following the Jotun Heim algorithm with the default settings. Second, the number of matched positions between the two aligned nucleic acid sequences is determined. A matched position refers to a position in which identical bases occur at the same position as aligned by the MEGALIGN(copyright) sequence alignment software. Third, the number of matched positions is divided by the total number of bases in the template sequence, and the resulting value multiplied by 100 to obtain the percent identity. If the obtained percent identity is greater than or equal to about 60 percent for a particular nucleic acid sequence, then that particular nucleic acid sequence is a nucleic acid sequence at least about 60 percent identical to a sequence set forth in the IEG nucleic acid group.
Any isolated nucleic acid having a nucleic acid sequence that encodes an amino acid sequence at least about 60 percent identical (e.g., at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent identical) to the sequence set forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62 is within the scope of the invention. For convenience, the amino acid sequences set forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, and 62 will be referred to collectively as the IEG amino acid group. For the purpose of this invention, the percent identity between a sequence set forth in the IEG amino acid group (designated a template sequence) and any other amino acid sequence is calculated as follows. First, the two amino acid sequences are aligned using the MEGALIGN(copyright) (DNASTAR, Madison, Wis., 1997) sequence alignment software following the Jotun Heim algorithm with the default settings. Second, the number of matched positions between the two aligned amino acid sequences is determined. A matched position refers to a position in which identical residues occur at the same position as aligned by the MEGALIGN(copyright) sequence alignment software. Third, the number of matched positions is divided by the total number of amino acid residues in the template sequence, and the resulting value multiplied by 100 to obtain the percent identity. If the obtained percent identity is greater than or equal to about 60 percent for a particular amino acid sequence, then that particular amino acid sequence is an amino acid sequence at least about 60 percent identical to a sequence set forth in the IEG amino acid group.
Any isolated nucleic acid having a nucleic acid sequence that encodes an amino acid sequence at least five amino acids in length also is within the scope of the invention provided the encoded amino acid sequence has at least three different amino acid residues, and is identical to a contiguous portion of sequence set forth in a sequence within the IEG amino acid group.
Further, any isolated nucleic acid having at least one adenine base, at least one guanine base, at least one cytosine base, and at least one thymine or uracil base is within the scope of the invention provided the isolated nucleic acid is at least about 12 bases in length (e.g., at least about 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 bases in length), and hybridizes, under hybridization conditions, to the sense or antisense strand of a nucleic acid having a sequence as set forth in the IEG nucleic acid group. The hybridization conditions can be moderately or highly stringent hybridization conditions.
For the purpose of this invention, moderately stringent hybridization conditions mean the hybridization is performed at about 42xc2x0 C. in a hybridization solution containing 25 mM KPO4 (pH7.4), 5xc3x97SSC, 5xc3x97Denharts solution, 50 xcexcg/ml denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/ml probe ( greater than 5xc3x97107 cpm/xcexcg), while the washes are performed at about 50xc2x0 C. with a wash solution containing 2xc3x97SSC and 0.1% SDS.
Highly stringent hybridization conditions mean the hybridization is performed at about 42xc2x0 C. in a hybridization solution containing 25 mM KPO4 (pH7.4), 5xc3x97SSC, 5xc3x97Denharts solution, 50 xcexcg/ml denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/ml probe ( greater than 5xc3x97107 cpm/xcexcg), while the washes are performed at about 65xc2x0 C. with a wash solution containing 0.2xc3x97SSC and 0.1% SDS.
Nucleic acid within the scope of the invention can be identified and obtained using any method including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, PCR can be used to obtain a nucleic acid having a nucleic acid sequence at least about 60 percent identical (e.g., at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent identical) to a sequence set forth in the IEG nucleic acid group. PCR refers to a procedure or technique in which target nucleic acid is amplified in a manner similar to that described in U.S. Pat. No. 4,683,195, and subsequent modifications of the procedure described therein. Generally, sequence information from the ends of the region of interest or beyond are used to design oligonucleotide primers that are identical or similar in sequence to opposite strands of a potential template to be amplified. Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. For example, a nucleic acid sequence can be isolated by PCR amplification from total cellular RNA, total genomic DNA, and cDNA as well as from bacteriophage sequences, plasmid sequences, viral sequences, and the like. When using RNA as a source of template, reverse transcriptase can be used to synthesize complimentary DNA strands.
Nucleic acid within the scope of the invention also can be obtained by mutagenesis. For example, a nucleic acid sequence set forth in the IEG nucleic acid group can be mutated using common molecular cloning techniques (e.g., site-directed mutageneses). Possible mutations include, without limitation, deletions, insertions, and base substitutions, as well as combinations of deletions, insertions, and base substitutions.
In addition, nucleic acid and amino acid databases (e.g., GenBank(copyright)) can be used to identify and obtain a nucleic acid within the scope of the invention. For example, any nucleic acid sequence having some homology to a sequence set forth in the IEG nucleic acid group, or any amino acid sequence having some homology to a sequence set forth in the IEG amino acid group can be used as a query to search GenBank(copyright).
Further, nucleic acid hybridization techniques can be used to identify and obtain a nucleic acid within the scope of the invention. Briefly, any nucleic acid having some homology to a sequence set forth in the IEG nucleic acid group, or fragment thereof, can be used as a probe to identify a similar nucleic acid by hybridization under conditions of moderate to high stringency. Such similar nucleic acid then can be isolated, sequenced, and analyzed to determine whether they are within the scope of the invention as described herein.
Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, that hybridizes to a probe. The probe can be labeled with a radioisotope such as 32P, an enzyme, digoxygenin, or by biotinylation. The DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory, Plainview, N.Y. Typically, a probe is at least about 20 nucleotides in length. For example, a probe corresponding to a 20 nucleotide sequence set forth in the IEG amino acid group can be used to identify a nucleic acid identical to or similar to a nucleic acid sequence set forth in the IEG nucleic acid group. In addition, probes longer or shorter than 20 nucleotides can be used.
Any cell containing an isolated nucleic acid within the scope of the invention is itself within the scope of the invention. This includes, without limitation, prokaryotic and eukaryotic cells. It is noted that cells containing an isolated nucleic acid of the invention are not required to express the isolated nucleic acid. In addition, the isolated nucleic acid can be integrated into the genome of the cell or maintained in an episomal state. In other words, cells can be stably or transiently transfected with an isolated nucleic acid of the invention.
Any method can be used to introduce an isolated nucleic acid into a cell. In fact, many methods for introducing nucleic acid into a cell, whether in vivo or in vitro, are well known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods that can be used to introduce nucleic acid into a cell. In addition, naked DNA can be delivered directly to cells in vivo as describe elsewhere (U.S. Pat. Nos. 5,580,859 and 5,589,466 including continuations thereof). Further, nucleic acid can be introduced into cells by generating transgenic animals.
Transgenic animals can be aquatic animals (such as fish, sharks, dolphin, and the like), farm animals (such as pigs, goats, sheep, cows, horses, rabbits, and the like), rodents (such as rats, guinea pigs, and mice), non-human primates (such as baboon, monkeys, and chimpanzees), and domestic animals (such as dogs and cats). Several techniques known in the art can be used to introduce nucleic acid into animals to produce the founder lines of transgenic animals. Such techniques include, without limitation, pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA, 82:6148-6152 (1985)); gene transfection into embryonic stem cells (Gossler A et al., Proc Natl Acad Sci USA 83:9065-9069 (1986)); gene targeting into embryonic stem cells (Thompson et al., Cell, 56:313-321 (1989)); nuclear transfer of somatic nuclei (Schnieke AE et al., Science 278:2130-2133 (1997)); and electroporation of embryos.
For a review of techniques that can be used to generate and assess transgenic animals, skilled artisans can consult Gordon (Intl. Rev. Cytol., 115:171-229 (1989)), and may obtain additional guidance from, for example: Hogan et al., xe2x80x9cManipulating the Mouse Embryoxe2x80x9d Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1986); Krimpenfort et al., Bio/Technology, 9:844-847 (1991); Palmiter et al., Cell, 41:343-345 (1985); Kraemer et al., xe2x80x9cGenetic Manipulation of the Early Mammalian Embryoxe2x80x9d Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1985); Hammer et al., Nature, 315:680-683 (1985); Purscel et al., Science, 244:1281-1288 (1986); Wagner et al., U.S. Pat. No. 5,175,385; and Krimpenfort et al., U.S. Pat. No. 5,175,384.
Any method can be used to identify cells that contain an isolated nucleic acid within the scope of the invention. For example, PCR and nucleic acid hybridization techniques such as Northern and Southern analysis can be used. In some cases, immunohistochemistry and biochemical techniques can be used to determine if a cell contains a particular nucleic acid by detecting the expression of a polypeptide encoded by that particular nucleic acid. For example, detection of polypeptide X-immunoreactivity after introduction of an isolated nucleic acid containing a cDNA that encodes polypeptide X into a cell that does not normally express polypeptide X can indicate that that cell not only contains the introduced nucleic acid but also expresses the encoded polypeptide X from that introduced nucleic acid. In this case, the detection of any enzymatic activities of polypeptide X also can indicate that that cell contains the introduced nucleic acid and expresses the encoded polypeptide X from that introduced nucleic acid.
In addition, any method can be used to force a cell to express an encoded amino acid sequence from a nucleic acid. Such methods are well known to those skilled in the art, and include, without limitation, constructing a nucleic acid such that a regulatory element drives the expression of a nucleic acid sequence that encodes a polypeptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. Such regulatory elements include, without limitation, promoters, enhancers, and the like. Further, any methods can be used to identifying cells that express an amino acid sequence from a nucleic acid. Such methods are well known to those skilled in the art, and include, without limitation, immunocytochemistry, Northern analysis, and RT-PCR.
The term xe2x80x9csubstantially purexe2x80x9d as used herein with reference to a polypeptide means the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that is removed from its natural environment and is at least 60 percent free, preferably 75 percent free, and most preferably 90 percent free from other components with which it is naturally associated. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
Any substantially pure polypeptide having an amino acid sequence encoded by a nucleic acid within the scope of the invention is itself within the scope of the invention. In addition, any substantially pure polypeptide having an amino acid sequence at least about 60 percent (e.g., at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent) identical to a sequence set forth in the IEG amino acid group is within the scope of the invention. The percent identity between particular amino acid sequences is determined as described herein.
Any method can be used to obtain a substantially pure polypeptide. For example, one skilled in the art can use common polypeptide purification techniques such as affinity chromotography and HPLC as well as polypeptide synthesis techniques. In addition, any material can be used as a source to obtain a substantially pure polypeptide. For example, tissue from wild-type or transgenic animals can be used as a source material. In addition, tissue culture cells engineered to overexpress a particular polypeptide of interest can be used to obtain substantially pure polypeptide. Further, a polypeptide within the scope of the invention can be xe2x80x9cengineeredxe2x80x9d to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag(copyright) tag (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini. Other fusions that could be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.
The term xe2x80x9cantibodyxe2x80x9d as used herein refers to intact antibodies as well as antibody fragments that retain some ability to selectively bind an epitope. Such fragments include, without limitation, Fab, F(abxe2x80x2)2, and Fv antibody fragments. The term xe2x80x9cepitopexe2x80x9d refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules (e.g., amino acid or sugar residues) and usually have specific three dimensional structural characteristics as well as specific charge characteristics.
Any antibody having specific binding affinity for an amino acid sequence encoded by a nucleic acid within the scope of the invention is itself within the scope of the invention. Thus, any monoclonal or polyclonal antibody having specific binding affinity for an amino acid sequence set forth in the IEG amino acid group is within the scope of the invention. Such antibodies can be used in immunoassays in liquid phase or bound to a solid phase. For example, the antibodies of the invention can be used in competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays include the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
Antibodies within the scope of the invention can be prepared using any method. For example, any substantially pure polypeptide provided herein, or fragment thereof, can be used as an immunogen to elicit an immune response in an animal such that specific antibodies are produced. Thus, an intact full-length polypeptide or fragments containing small peptides can be used as an immunizing antigen. In addition, the immunogen used to immunize an animal can be chemically synthesized or derived from translated cDNA. Further, the immunogen can be conjugated to a carrier polypeptide, if desired. Commonly used carriers that are chemically coupled to an immunizing polypeptide include, without limitation, keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
The preparation of polyclonal antibodies is well-known to those skilled in the art. See, e.g., Green et al., Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press 1992) and Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992). In addition, those of skill in the art will know of various techniques common in the immunology arts for purification and concentration of polyclonal antibodies, as well as monoclonal antibodies (Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).
The preparation of monoclonal antibodies also is well-known to those skilled in the art. See, e.g., Kohler and Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor Pub. 1988). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by analyzing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG), in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana Press 1992).
In addition, methods of in vitro and in vivo multiplication of monoclonal antibodies is well-known to those skilled in the art. Multiplication in vitro can be carried out in suitable culture media such as Dulbecco""s Modified Eagle Medium or RPMI 1640 medium, optionally replenished by mammalian serum such as fetal calf serum, or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, and bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells (e.g., osyngeneic mice) to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
The antibodies within the scope of the invention also can be made using non-human primates. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO 91/11465 (1991) and Losman et al., Int. J. Cancer 46:310 (1990).
Alternatively, the antibodies can be xe2x80x9chumanizedxe2x80x9d monoclonal antibodies. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions (CDRs) from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions when treating humans. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Nat""l. Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Carter et al., Proc. Nat""l. Acad. Sci. USA 89:4285 (1992); Sandhu, Crit. Rev. Biotech. 12:437 (1992); and Singer et al., J. Immunol. 150:2844 (1993).
Antibodies of the present invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991) and Winter et al., Ann. Rev. Immunol. 12: 433 (1994). Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been xe2x80x9cengineeredxe2x80x9d to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens and can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579 (1994).
Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of an intact antibody or by the expression of a nucleic acid encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of intact antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(abxe2x80x2)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fabxe2x80x2 monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fabxe2x80x2 fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg (U.S. Pat. Nos. 4,036,945 and 4,331,647). See also Nisonhoff et al., Arch. Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman et al., METHODS IN ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used provided the fragments retain some ability to selectively bind its epitope.
For example, Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat""l. Acad. Sci. USA 69:2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding polypeptides (sFv) are prepared by constructing a nucleic acid construct encoding the VH and VL domains connected by an oligonucleotide. This nucleic acid construct is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird et al., Science 242:423-426 (1988); Ladner et al., U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology 11:1271-77 (1993); and Sandhu, supra.
Another form of an antibody fragment is a peptide coding for a single CDR. CDR peptides (xe2x80x9cminimal recognition unitsxe2x80x9d) can be obtained by constructing nucleic acid constructs that encode the CDR of an antibody of interest. Such constructs are prepared, for example, by using PCR to synthesize the variable region from RNA of antibody-producing cells. See, e.g., Larrick et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).
It is also possible to use anti-idiotype technology to produce monoclonal antibodies that mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the xe2x80x9cimagexe2x80x9d of the epitope bound by the first monoclonal antibody. Such anti-idiotypic monoclonal antibodies can be used to inhibit the activity of the polypeptide containing the original epitope.
The invention also provides cDNA libraries enriched for IEGs. As described herein, such cDNA libraries contain an increased frequency of cDNAs derived from IEGs. Specifically, about 15 percent (e.g., about 20 or 25 percent) of the cDNA clones within the cDNA libraries provided herein are derived from IEGs.
A cDNA library within the scope of the invention can be prepared from any tissue containing cells that express an IEG (e.g., hippocampus tissue). Again, an IEG is a gene whose expression is rapidly increased immediately following a stimulus. The stimulus can be electrical or chemical in nature. For example, cells can be treated with electric shock or chemicals such as kainate. Briefly, cDNA libraries are prepared from the hippocampus of control animals (e.g., rats) as well as from animals that receive a stimulus (e.g., multiple MECS) using, for example, a phage vector lambda ZAP II (Stratagene). A subtracted library is then prepared using in vitro mRNA prepared from a control library and subsequent solution phase hybridization with cDNA prepared from a stimulated library. The control in vitro mRNA can be tagged with biotin to permit its removal from solution using avidin beads (Lanahan et al., Mol. Cell. Biol. 12:3919-3929 (1992)). cDNA that remains after removal of mRNA/cDNA hybrids can be recloned into, for example, a lambda ZAPII phage vector. Several rounds of subtraction (e.g., two, three, four, or five rounds) can be used to increase the frequency of IEGs. The subtracted library then can be plated and duplicate phage lifts screened with a radiolabeled cDNA probe. Any probe can be used provided it contains at least one nucleic acid sequence derived from an IEG. For example, a probe can be prepared from mRNA obtained from the hippocampus of a stimulated animal. In addition, the mRNA used to make a probe can be subjected to subtractive hybridization such that IEG sequences are enriched. In general, conventional cDNA libraries contain IEGs at a frequency of  less than 1:30,000 cDNAs. For the cDNA libraries enriched for IEGs, however, about 1 in 5 genes can be induced by a stimulus such as MECS. This represents an about 1000 to 10,000 fold enrichment in brain IEGs.
An animal (e.g., human) having a deficiency in a neuron""s IEG responsiveness to a stimulus (e.g., a stimulus that influences learning or memory) can be treated using the methods and materials described herein. A stimulus that influences learning or memory can be a multiple MECS treatment. A deficiency in a neuron""s IEG responsiveness to a stimulus means the level of IEG responsiveness is not normal. Such deficiencies can be identified by stimulating a sample of cells and measuring the levels of IEG expression. If the levels are not similar to the levels normally observed in a similar tissue sample, then there is a deficiency. It is noted that increased IEG expression as well as decreased IEG expression can be classified as a deficiency provided the levels are not normal.
A deficiency in a neuron""s IEG responsiveness to a stimulus can be treated by administering a nucleic acid of the invention to the animal such that the effect of the deficiency is minimized. The administration can be an in vivo, in vitro, or ex vivo administration as described herein. For example, an in vivo administration can involve administering a viral vector to the hippocampal region of an animal, while an ex vivo administration can involve extracting cells from an animal, transfecting the cells with the nucleic acid in tissue culture, and then introducing the transfected cells back into the same animal.
In addition, a deficiency in a neuron""s IEG responsiveness to a stimulus can be treated by administering a therapeutically effective amount cells containing isolated IEG nucleic acid, substantially pure IEG polypeptides, anti-IEG polypeptide antibodies, or combinations thereof. A therapeutically effective amount is any amount that minimizes the effect of the deficiency while not causing significant toxicity to the animal. Such an amount can be determined by assessing the clinical symptoms associated with the deficiency before and after administering a fixed amount of cells, polypeptides, or antibodies. In addition, the effective amount administered to an animal can be adjusted according to the animal""s response and desired outcomes. Significant toxicity can vary for each particular patient and depends on multiple factors including, without limitation, the patient""s physical and mental state, age, and tolerance to pain. The cells, polypeptides, or antibodies can be administered to any part of the animal""s body including, without limitation, brain, spinal cord, bloodstream, muscle tissue, skin, peritoneal cavity, and the like. Thus, these therapeutic agents can be administered by injection (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, intracavity, or transdermal injection) or by gradual perfusion over time.
Preparations for administration can include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Other vehicles for adminstration include sodium chloride solution, Ringer""s dextrose, dextrose and sodium chloride, lactated Ringer""s intravenous vehicles containing fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer""s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Further, a deficiency in a neuron""s IEG responsiveness to a stimulus can be treated by administering a therapeutically effective amount of a compound that directly interferes with the translation of IEG nucleic acid. For example, antisense nucleic acid or ribozymes could be used to bind to IEG mRNA or to cleave it. Antisense RNA or DNA molecules bind specifically with a targeted RNA message, interrupting the expression of the mRNA product. The antisense binds to the messenger RNA forming a double stranded molecule that cannot be translated by the cell. Typically, an antisense oligonucleotides is about 15-25 nucleotides in length. In addition, chemically reactive groups, such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe), can be attached to an antisense oligonucleotide, causing cleavage of the mRNA at the site of hybridization. These and other uses of antisense methods to inhibit the translation of nucleic acid are well known in the art (Marcus-Sakura, Anal. Biochem., 172:289 (1988)).
An oligonucleotide also can be used to stall transcription winding around double-helical DNA and forming a three-strand helix (Maher, et al., Antisense Res. and Dev., 1:227 (1991) and Helene, Anticancer Drug Design, 6:569 (1991)).
Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. By modifying nucleic acid sequences that encode ribozymes, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030 (1988)). There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature, 334:585 (1988)) and xe2x80x9chammerheadxe2x80x9d-type. Tetrahymena-type ribozymes recognize sequences that are four bases in length, while xe2x80x9chammerheadxe2x80x9d-type ribozymes recognize sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, xe2x80x9chammerheadxe2x80x9d-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species. In addition, 18-based recognition sequences are preferable to shorter recognition sequences. These and other uses of antisense methods to inhibit the in vivo translation of nucleic acid are well known in the art (DeMesmaeker et al., Curr. Opin. Struct. Biol. 5:343-355 (1995); Gewirtz et al., Proc. Nat""l. Acad. Sci. U.S.A., 93:3161-3163 (1996); and Stein, Chem. Biol. 3:319-323 (1996)).
Delivery of nucleic acid, antisense, triplex agents, and ribozymes can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Various viral vectors that can be utilized for gene therapy include adenoviruses, herpesviruses, vaccinia viruses, and retroviruses. A retroviral vector can be a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. In addition, a nucleic acid sequence of interest along with another nucleic acid sequence that encodes a ligand for a receptor on a specific target cell can be inserted into a viral vector to produce a vector that is target specific. For example, retroviral vectors can be made target specific by inserting a nucleic acid sequence that encodes an antibody that binds a specific target antigen. Those of skill in the art can readily ascertain without undue experimentation specific nucleic acid sequences that can be inserted into a retroviral genome to allow target specific delivery of the retroviral vector containing the nucleic acid of the invention.
A colloidal dispersion system can be used to target the delivery of the nucleic acid of the invention. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV) that range in size from 0.2-4.0 xcexcm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. Thus, nucleic acid, intact virions, polypeptides, and antibodies can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al., Trends Biochem. Sci., 6:77 (1981)). In addition to mammalian cells, liposomes have been used to deliver nucleic acid to plants, yeast, and bacteria. In order for a liposome to be an efficient nucleic acid transfer vehicle, the following characteristics should be present: (1) encapsulation of the nucleic acid of interest at high efficiency while not compromising its biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of the nucleic acid (Mannino et al., Biotechniques, 6:682 (1988).
The composition of a liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids also can be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. In general, the compounds bound to the surface of the targeted delivery system will be ligands and receptors that allow the targeted delivery system to find and xe2x80x9chome inxe2x80x9d on the desired cells. A ligand may be any compound of interest that will bind to another compound, such as a receptor or antibody.
Compounds that modulate IEG expression can be identified by contacting a test compound with an IEG nucleic acid, and determining whether the test compound effects expression. Likewise, compounds that modulate IEG polypeptide activity can be identified by contacting a test compound with an IEG polypeptide, and determining whether the test compound effects polypeptide activity. Contacting includes in solution and in solid phase, or in a cell. Any type of compound can be used as a test compound including, without limitation, peptides, peptidomimetics, polypeptides, chemical compounds, and biologic agents. In addition, the test compound can be a combinatorial library for screening a plurality of compounds. Compounds identified using the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et al., Proc. Nat""l. Acad. Sci. USA, 80:278 (1983), oligonucleotide ligation assays (OLAs; Landegren, et al., Science, 241:1077 (1988), and the like.